T cell activation is central to initiating an immune response. Minimally, two signals are required: specific recognition of antigen through the T cell receptor (TCR) and a co-stimulatory signal, primarily provided by CD28 in na[unreadable]ve T cells. TCR activation without appropriate co-stimulation not only fails to activate T cells but also leads to a state of unresponsiveness, or anergy. Therapeutic inhibition of inappropriate T cell activation by co-stimulatory modulation is currently under investigation for several autoimmune diseases. Although the importance of CD28 and co-stimulatory signals in T cell activation and the immune response is understood, the biochemical signals resulting from CD28 engagement on the cell surface are poorly characterized. Therefore, to identify novel molecules that regulate T cell activation, but which may function outside of well-characterized TCR signaling pathways, we established a unique system coupling genetic complementation with Jurkat mutagenesis. Using the RE/AP reporter element as a model of signal integration, we generated mutant clones in the Jurkat T cell line in which TCR-mediated signaling leading to NFAT activation is normal, while co-stimulation leading to IL-2 upregulation is abrogated. Biochemical characterization of these cell lines failed to pinpoint the molecular cause for the defects in T cell activation in these cell lines, implying that novel molecules or pathways may be responsible. Therefore, we took a genetic approach to rescue the T cell activation defect by retroviral expression of a leukocyte library. From our initial screen, we identified one cell line in which T cell activation was restored due to overexpression of a poorly characterized protein, NKAP. Subsequent work has identified NKAP as a component of the Notch co-repressor complex. Since Notch signaling has been shown to regulate T cell activation and IL-2 production, it demonstrates a proof-of-principal that this genetic screen is capable of identifying novel regulators of T cell activation. Thus, we have established a functional screen that will lead to novel insights into the biochemical regulation of T cell activation, and mining this system will likely lead to additional molecules critical to generating an immune response. Our specific aims are: Specific Aim #1. Genetic complementation of Jurkat mutant cell lines via retroviral transduction of a cDNA utilizing a second-generation dual readout. Specific Aim #2. Biochemical characterization of the function of novel regulators of T cell activation identified by the complementation screen. PUBLIC HEALTH RELEVANCE: Biochemical pathways initiated upon T cell activation regulate the immune system responses to either a foreign pathogen, or to itself as occurs in autoimmune disease. Manipulation of these pathways is currently under investigation for their use in immunotherapy. This proposal focuses on identifying novel molecules that regulate T cell activation, which may become good targeted for therapeutic intervention.